Process for producing poxviruses and poxvirus compositions

ABSTRACT

The present invention relates to compositions and pharmaceutical compositions comprising poxviruses and more particularly extracellular enveloped viruses. The present invention also relates to a process for producing poxviruses and poxviruses obtained thereof. Moreover, the present invention also relates to the use of said poxvirus and said composition for the preparation of a medicament.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Continuation application of U.S. patentapplication Ser. No. 12/304,353, filed on May 18, 2009, now U.S. Pat.No. 8,058,049, which is a U.S. National Stage pursuant to 35 U.S.C. §371of International Patent Application PCT/EP2007/005302, filed on Jun. 15,2007, and published as WO 2007/147528 on Dec. 27, 2007, which claimspriority to U.S. Provisional Patent Application Ser. No. 60/861,452,filed on Nov. 29, 2006, and EP 06360027.4, filed on Jun. 20, 2006, allof which are incorporated herein by reference in their entireties forall purposes.

The present invention relates to compositions and pharmaceuticalcompositions comprising poxviruses and more particularly extracellularenveloped viruses. The present invention also relates to a process forproducing poxviruses and poxviruses obtained thereof. Moreover, thepresent invention also relates to the use of said poxvirus and saidcomposition for the preparation of a medicament.

The arising of new threats (avian flu, west nile virus, anthrax, etc. .. . ) as well as the development of gene therapy has increased the needfor producing and purifying poxviruses for prophylactic or therapeuticpurposes. This is notably the case for the Mammalian Virus Ankara (MVA).This poxvirus which was initially used for vaccinating immunodeficientpatients against Variola, is now also used as a vector for gene therapypurposes. For example, MVA is utilized as a vector for the MUC1 gene forvaccinating patients against tumor expressing Muc1 (Scholl et al., 2003,J Biomed Biotechnol., 2003, 3, 194-201). MVA carrying the gene codingHPV antigens is also used as a vector for the therapeutic treatment ofovarian carcinoma.

Poxviruses are a group of complex enveloped viruses that distinguishthem principally by their unusual morphology, their large DNA genome andtheir cytoplasmic site of replication. The genome of several members ofpoxyiridae, including the Copenhagen vaccinia virus (VV) strain (Goebelet al., 1990, Virol. 179, 247-266 and 517-563; Johnson et al., 1993,Virol. 196, 381-401) and the modified vaccinia virus Ankara (MVA) strain(Antoine et al., 1998, Virol. 244, 365-396), have been mapped andsequenced. VV has a double-stranded DNA genome of about 192 kb codingfor about 200 proteins of which approximately 100 are involved in virusassembly. MVA is a highly attenuated vaccinia virus strain generated bymore than 500 serial passages of the Ankara strain of vaccinia virus onchicken embryo fibroblasts (Mayr et al., 1975, Infection 3, 6-16). TheMVA virus was deposited before Collection Nationale de Cultures deMicroorganismes (CNCM) under depositary N⁶⁰² I-721. Determination of thecomplete sequence of the MVA genome and comparison with the CopenhagenVV genome allows the precise identification of the alterations whichoccurred in the viral genome and the definition of seven deletions (I toVII) and numerous mutations leading to fragmented ORFS (Open ReadingFrame) (Antoine et al., 1998, Virology 244, 365-396).

The natural pathway for intracellular uptake of enveloped virusesinvolves a series of steps including the binding of a viral polypeptideexposed at the virus surface to a cellular receptor and a fusionmechanism between the viral and cellular membranes resulting in viralgenome release into the cytoplasm of the infected cell.

However, in poxvirus special case, the exact delivery pathway analysisis complicated by the existence of two morphologically distinct forms ofinfectious virus, termed intracellular mature virus (IMV) andextracellular enveloped virus (EEV). The IMV form is, among otherparticularities, characterized by a monolipid envelope surrounding theviral core and is principally localized in the cytoplasm of the infectedcells, although it might be extracellularly released after lysis of theinfected cells. Many of the natural polypeptides exposed at the surfaceof the IMV lipid envelope have been identified, such as for example thep14 kDa and p21 kDa proteins, respectively encoded by the A27L gene(Rodriguez at al., 1985, J. Virol. 56, 482-488; Rodriguez et Estaban,1987, J. Virol. 61, 3550-3554) and the A17L gene, as well as proteinsencoded by L1R, A14L, D8L, A9L (Yeh et al., 2000, J. Virol. 74,9701-9711), E10R (Senkevich et al., 2000, Virol. 5, 244-252) and H3Lgenes. Compared to the IMV, the EEV form has an additional outer lipidmembrane envelope (double lipid layer) acquired from the trans-Golginetwork cisternae. It corresponds to the viral form released outside theinfected cells. The EEV surface membrane envelope shows about 10proteins which are absent from the IMV surface, such as for example theencoded B5R, A34R and hemagglutinin (HA) gene products. The co-existenceof said IMV and EEV forms has been described for most of the vacciniastrains (e.g. Copenhagen and MVA strains) as well as for otherpoxviruses such as the fowl poxvirus (Boulanger et al., 2000, J. Gen.Virol. 81, 675-687).

As they are most stable in the environment, IMVs play a predominant rolein a host to host transmission (Hooper et al. Virology, 2003, 306,181-185). With this respect, IMV particles have been the vectors ofchoice for gene therapy purposes. For this reason, the availablepoxvirus purification processes only treat the virus present in thepackaging cells (i.e. IMV), whereas the EEV particles shed into theculture media are discarded. Because of the presence at its surface of alarger variety of polypeptides than onto the IMV surface, the use ofrecombinant EEVs having a targeted infection specificity has alreadybeen proposed (US20050208074; Galmiche et al. J. Gen. Virol., 1997, 78,3019-3027). However, even for this specific use, IMV particles areparticularly preferred (US20050208074, page 4, chapter 29).

Surprisingly, the applicant has found that EEVs with no targetedinfection specificity have a greater therapeutic and/or prophylacticefficacy compared to IMV.

With this respect, the present invention relates to a poxvirus andpreferably a recombinant poxvirus, wherein said poxvirus is an EEV withno targeted infection specificity. The present invention also relates toa composition and preferably a pharmaceutical composition comprisingrecombinant EEV with no targeted infection specificity.

As used throughout the entire application, the terms “a” and “an” areused in the sense that they mean “at least one”, “at least a first”,“one or more” or “a plurality” of the referenced components or steps,unless the context clearly dictates otherwise. For example, the term “acell” includes a plurality of cells, including mixtures thereof.

The term “and/or” wherever used herein includes the meaning of “and”,“or” and “all or any other combination of the elements connected by saidterm”.

The term “about” or “approximately” as used herein means within 20%,preferably within 10%, and more preferably within 5% of a given value orrange.

As used herein, the term “comprising” is intended to mean that theproducts, compositions and methods include the referenced components orsteps, but not excluding others. “Consisting essentially of” when usedto define products, compositions and methods, shall mean excluding othercomponents or steps of any essential significance. Thus, a compositionconsisting essentially of the recited components would not exclude tracecontaminants and pharmaceutically acceptable carriers. “Consisting of”shall mean excluding more than trace elements of other components orsteps.

Poxvirus family comprises viruses of the Chordopoxvirus andEntomopoxvirus subfamilies. Among these, the poxvirus according to theinvention is preferably chosen from the group comprisingOrthopoxviruses, Parapoxviruses, Avipoxviruses, Capripoxviruses,Leporipoxviruses, Suipoxviruses, Molluscipoxyi ruses, Yatapoxviruses.According to a more preferred embodiment, the poxvirus of the inventionis an orthopoxvirus.

The Orthopoxvirus is preferably a vaccinia virus and more preferably amodified vaccinia virus Ankara (MVA) in particular MVA 575 (ECACCV00120707) and MVA-BN (ECACC V00083008).

As previously indicated, an IMV particle comprises the viral coreincluding the viral genome surrounded by a monolayer lipid envelope. Theterm “EEV” refers to an IMV particle surrounded by an additional bilayerlipid envelope exposing at its surface cellular as well as viralpolypeptides.

The term “targeted infection specificity” as used herein refers to acontrolled infection specificity, where a poxyiral particle isengineered to display a new or enhanced tropism towards a target cell,compared to a related non modified poxvirus particle. As a result, apoxyiral particle with a targeted infection specificity shows apropensity to infect said target cells unlike its related non modifiedpoxyiral particle, which means that the poxyiral particle with atargeted infection specificity infects more efficiently or more rapidelyits target cells (displaying at their surface the anti-ligand recognizedby the ligand moiety displayed at the surface of the poxyiral particleof the invention) than non target cells (that do not display at theirsurface such an anti-ligand), whereas a related poxyiral particle withno targeted infection specificity will infect said target cells with alower or a similar efficiency compared to non-target cells.

The term “recombinant virus” refers to a virus comprising an exogenoussequence inserted in its genome. As used herein, an exogenous sequencerefers to a nucleic acid which is not naturally present in the parentvirus.

In one embodiment, the exogenous sequence encodes a molecule having adirectly or indirectly cytotoxic function. By “directly or indirectly”cytotoxic, we mean that the molecule encoded by the exogenous sequencemay itself be toxic (for example ricin, tumour necrosis factor,interleukin-2, interferon-gamma, ribonuclease, deoxyribonuclease,Pseudomonas exotoxin A) or it may be metabolised to form a toxicproduct, or it may act on something else to form a toxic product. Thesequence of ricin cDNA is disclosed in Lamb et al (Eur. J. Biochem.,1985, 148, 265-270) incorporated herein by reference.

In a preferred embodiment of the invention, the exogenous sequence is asuicide gene. A suicide gene encodes a protein able to convert arelatively non-toxic prodrug to a toxic drug. For example, the enzymecytosine deaminase converts 5-fluorocytosine (5FC) to 5-fluorouracil(5FU) (Mullen et al (1922) PNAS 89, 33); the herpes simplex enzymethymidine kinase sensitises cells to treatment with the antiviral agentganciclovir (GCV) or aciclovir (Moolten (1986) Cancer Res. 46, 5276;Ezzedine et al (1991) New Biol 3, 608). The cytosine deaminase of anyorganism, for example E. coli or Saccharomyces cerevisiae, may be used.

Thus, in a more preferred embodiment of the invention, the gene encodesa protein having a cytosine deaminase activity and even more preferablya protein as described in patent applications WO2005007857 andWO9954481.

Other examples of pro-drug/enzyme combinations include those disclosedby Bagshawe et al (WO88/07378), namely various alkylating agents and thePseudomonas spp. CPG2 enzyme, and those disclosed by Epenetos &Rowlinson-Busza (WO 91/11201), namely cyanogenic pro-drugs (for exampleamygdalin) and plant-derived beta-glucosidases.

Enzymes that are useful in this embodiment of the invention include, butare not limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; proteases, suchas serratia protease, thermolysin, subtilisin, carboxypeptidases andcathepsins (such as cathepsins B and L), that are useful for convertingpeptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases,useful for converting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as beta-galactosidase andneuraminidase useful for converting glycosylated prodrugs into freedrugs; beta-lactamase useful for converting drugs derivatized withbeta-lactams into free drugs; and penicillin amidases, such aspenicillin V amidase or penicillin G amidase, useful for convertingdrugs derivatized at their amine nitrogens with phenoxyacetyl orphenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as abzymes,can be used to convert the prodrugs of the invention into free activedrugs (Massey R. et al., Nature, 1987, 328, 457-458).

Similarly, prodrugs include, but are not limited to, the above-listedprodrugs, e.g., phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,beta-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted. Examples of cytotoxicdrugs that can be derivatized into a prodrug form for use in thisinvention include, but are not limited to, etoposide, teniposide,adriamycin, daunomycin, caminomycin, aminopterin, dactinomycin,mitomycins, cis-platinum and cis-platinum analogues, bleomycins,esperamicins (see for example U.S. Pat. No. 4,675,187), 5-fluorouracil,melphalan and other related nitrogen mustards.

In a further embodiment the exogenous gene encodes a ribozyme capable ofcleaving targeted RNA or DNA. The targeted RNA or DNA to be cleaved maybe RNA or DNA which is essential to the function of the cell andcleavage thereof results in cell death or the RNA or DNA to be cleavedmay be RNA or DNA which encodes an undesirable protein, for example anoncogene product, and cleavage of this RNA or DNA may prevent the cellfrom becoming cancerous.

In a still further embodiment the exogenous gene encodes an antisenseRNA.

By “antisense RNA” we mean an RNA molecule which hybridises to, andinterferes with the expression from a mRNA molecule encoding a proteinor to another RNA molecule within the cell such as pre-mRNA or tRNA orrRNA, or hybridises to, and interferes with the expression from a gene.

In another embodiment of the invention, the exogenous sequence replacesthe function of a defective gene in the target cell. There are severalthousand inherited genetic diseases of mammals, including humans, whichare caused by defective genes. Examples of such genetic diseases includecystic fibrosis, where there is known to be a mutation in the CFTR gene;Duchenne muscular dystrophy, where there is known to be a mutation inthe dystrophin gene; sickle cell disease, where there is known to be amutation in the HbA gene. Many types of cancer are caused by defectivegenes, especially protooncogenes, and tumour-suppressor genes that haveundergone mutation.

Examples of protooncogenes are ras, src, bcl and so on; examples oftumour-suppressor genes are p53 and Rb.

In a further embodiment of the invention, the exogenous sequence encodesa Tumor Associated Antigen (TAA). TAA refers to a molecule that isdetected at a higher frequency or density in tumor cells than innon-tumor cells of the same tissue type. Examples of TAA includes butare not limited to CEA, MART-1, MAGE-1, MAGE-3, GP-100, MUC-1, MUC-2,pointed mutated ras oncogene, normal or point mutated p53, overexpressedp53, CA-125, PSA, C-erb/B2, BRCA I, BRCA II, PSMA, tyrosinase, TRP-1,TRP-2, NY-ESO-1, TAG72, KSA, HER-2/neu, bcr-abl, pax3-fkhr, ews-fli-1,surviving and LRP. According to a more preferred embodiment the TAA isMUC1.

The recombinant poxvirus can comprise more than one exogenous sequenceand each exogenous sequence can encodes more than one molecule. Forexample, it can be useful to associate in a same recombinant poxvirus,an exogenous sequenced coding a TAA with an exogenous sequence coding acytokine.

In another embodiment of the invention, the exogenous gene encodes anantigen. As used herein, “antigen” refers to a ligand that can be boundby an antibody; an antigen need not itself be immunogenic.

Preferably the antigen is derived from a virus such as for exampleHIV-1, (such as gp120 or gp 160), any of Feline Immunodeficiency virus,human or animal herpes viruses, such as gD or derivatives thereof orImmediate Early protein such as ICP27 from HSV1 or HSV2, cytomegalovirus(such as gB or derivatives thereof), Varicella Zoster Virus (such asgpl, II or III), or from a hepatitis virus such as hepatitis B virus forexample Hepatitis B Surface antigen or a derivative thereof, hepatitis Avirus, hepatitis C virus (preferentially non structural protein fromgenotype 1b strain ja) and hepatitis E virus, or from other viralpathogens, such as Respiratory Syncytial Virus, Human Papilloma Virus(preferentially the E6 and E7 protein from the HPV16 strain) orInfluenza virus, or derived from bacterial pathogens such as Salmonella,Neisseria, Borrelia (for example OspA or OspB or derivatives thereof),or Chlamydia, or Bordetella for example P.69, PT and FHA, or derivedfrom parasites such as plasmodium or Toxoplasma.

In a particularly preferred embodiment of the invention, the recombinantpoxvirus encodes the same proteins than TG4010 (Rochlitz et al. J GeneMed. 2003 August; 5(8):690-9) and TG4001 (Liu et al. Proc Natl Acad SciUSA. 2004 Oct. 5; 101 Suppl 2:14567-71). In another particularlypreferred embodiment of the invention, the poxvirus of the invention hasa sequence which is more than 90% homologous to the sequence of TG4010or of TG4001.

Advantageously, the recombinant poxvirus further comprises the elementsnecessary for the expression of the exogenous sequence. The elementsnecessary for the expression comprise of the set of elements allowingthe transcription of a nucleotide sequence to RNA and the translation ofa mRNA to a polypeptide, in particular the promoter sequences and/orregulatory sequences which are effective in the cell to be infected bythe recombinant poxvirus of the invention, and optionally the sequencesrequired to allow the excretion or the expression at the surface of thecells for said polypeptide. These elements may be inducible orconstitutive. Of course, the promoter is adapted to the recombinantpoxvirus selected and to the host cell. There may be mentioned, by wayof example, the vaccinia virus promoters p7.5K pH5R, pK1L, p28, p11 or acombination of said promoters. The literature provides a large amount ofinformation relating to such promoter sequences.

The elements necessary may, in addition, include additional elementswhich improve the expression of the exogenous sequence or itsmaintenance in the host cell. There may be mentioned in particular theintron sequences (WO 94/29471), secretion signal sequences, nuclearlocalization sequences, internal sites for reinitiation of translationof the IRES type, poly A sequences for termination of transcription.

Available poxvirus production processes comprised the replication of thevirus in a cell line (e.g. HelaS3), in embryonated eggs or in ChickenEmbryo Fibroblasts. After the replication of the virus, the culturemedia is discard, the cells are lysed and the poxvirus released from thecells is purified by sucrose cushion centrifugation (Kotwal and Abraham;Poxvirus growth, Purification and tittering in Vaccinia Virus andPoxyirology, 2004, 101-108, Humana Press Inc., Totowa; NJ; USA). Withthese processes, no EEVs are present in the purified composition.

However, the available virus production processes are not satisfactory.Firstly, they comprise the use of compounds deriving from animals suchas serum and enzymes. The use of compounds deriving from animals inproduction processes has several drawbacks. For example, the chemicalcomposition of these compounds may vary between lots, even from a singlemanufacturer. The compounds may also be contaminated with infectiousagents (e.g., mycoplasma and viruses) which can seriously undermine thehealth of the cultured cells when these contaminated supplements areused in cell culture media formulations. Moreover, the use of serumsupplementation of culture media can complicate and increase the costsof the purification of the desired substances from the culture media dueto nonspecific co-purification of serum or extract proteins. Mostimportantly, the use of undefined compounds may hinder the approval bymedical agencies of the pharmaceutical composition obtained by theprocess.

The present invention further provides a process for producing apoxyiral particle according to the invention, comprising the steps of:

a) preparing a culture of packaging cells,

b) infecting said cell culture,

c) culturing said infected cells for an appropriate period of time,

d) recovering the poxyiral particles produced from the culturesupernatant and/or the packaging cells, and

e) optionally, purifying the recovered poxyiral particles.

The process according to the invention is preferably free from animalproducts.

The process according to the invention can also be used for theproduction of a wild type, and/or an attenuated poxvirus.

As used herein, the term “attenuated poxvirus” refers to any poxvirusthat has been modified so that its pathogenicity in the intended subjectis substantially reduced. Preferably, the poxvirus is attenuated to thepoint it is nonpathogenic from a clinical standpoint, i.e., thatsubjects exposed to the poxvirus do not exhibit a statisticallysignificant increased level of pathology relative to control subjects.According to a preferred embodiment of the invention, the attenuatedvirus is an attenuated Vaccinia virus such as MVA.

The term “infection” refers to the transfer of the viral nucleic acid toa cell, wherein the viral nucleic acid is replicated, viral proteins aresynthesized, or new viral particles assembled.

As used herein, the term “packaging cell” refers to a cell which can beinfected by the poxvirus to be produced. The packaging cell can be aprimary cell, a recombinant cell and/or a cell line. For example, arecombinant cell which contains the elements necessary for theproduction of a recombinant virus which are lacking in a recombinantviral vector can be used.

In one embodiment of the invention, the packaging cell is an immortalavian cell.

In one embodiment of the invention, the packaging cell is a DF1 cell(U.S. Pat. No. 5,879,924), which is a spontaneously immortalized chickencell line derived from 10 day old East Lansing Line (ELL-0) eggs.

Immortal avian cell can be derived from embryonic stem cells byprogressive severance from growth factors and feeder layer, thusmaintaining growth features and infinite lifespan characteristic ofundifferentiated stem). For example, the Ebx chicken cell line(WO2005007840) has been obtained by this process.

According to a preferred embodiment, a duck embryo permanent cell linecan also be used. For example, the cell line, designated as DEC 99(Ivanov et al. Experimental Pathology And Parasitology, 4/2000 BulgarianAcademy of Sciences) has been cultured over 140 consecutive passages andit is not tumorogenic for birds. The DEC 99 cell line is a standard cellculture system that has been used for research and can be applied forthe needs of biotechnology. According to a more preferred embodiment,the packaging cell is a cell line obtained by the process disclosed inpatent application EP06360001.9.

According to another preferred embodiment, the packaging cell used inthe process according to the invention is a chicken embryo fibroblast(CEF). The preparation and use of CEF for the production of viruses arewell known to the one skilled in the art.

CEF are preferably extracted from Specific Pathogen Free (SPF) eggs. SPFeggs are commercially available, for example from Charles RiverLaboratories (Wilmington, Mass., USA). Said eggs are preferably morethan 9 days old, more preferably between, 10 and 14 days old and evenmore preferably are 12 days old.

Before the extraction of the embryo, the egg is preferably disinfected.Many methods and products dedicated to the disinfection of eggs areavailable in the prior art. Incubation in a formol solution (e.g. 2%formol, 1 min.) followed by a rinsing in 70% ethanol is particularlypreferred.

The cells of the embryos are then dissociated and purified. According toa preferred embodiment of the invention, the cells are subjected to anenzymatic digestion step that allows the destruction of theintercellular matrix. For this purpose, the use of enzyme able to digestthe intercellular matrix is particularly useful. Such enzyme can beselected from the group comprising but not limited to Trypsin,Collagenase, Pronase, Dispase, Hyaluronidase and Neuraminidase. Thisenzyme can be used alone or in combination. In a particularly preferredembodiment of the invention dispase and Tryspsin (e.g. TrypLE selectfrom Gibco™) are used in combination. The one skilled in the art is ableto determine the enzyme concentration, the temperature and the length ofincubation allowing an efficient separation of the cells.

According to a preferred embodiment, the process according to theinvention is free from animal products (except the packaging cell). Withthis respect, the enzyme(s) used for the preparation of CEF is (are)preferably of recombinant origin. As used herein, <<animal products>>means any compound or collection of compounds that was produced in or byan animal cell in a living organism.

The preparation of CEF can further includes a filtration step (and/or acentrifugation step in order to remove contaminants.

This primary CEF cell can either be used directly or after one furthercell passage as secondary CEF cell.

The one skilled in the art is able to select the most appropriate cellfor the production of a specific virus. According to a preferredembodiment, the process according to the invention comprises the use ofCEF or a cell according to EP06360001.9 for the production of a MVA.

The packaging cell used in the process according to the invention iscultivated in an appropriate cell culture media. It is possible to usemore than one culture medium in the process according to the invention.For example, a first culture medium can be used during the preparationof the packaging cell (i.e. during step a) and a second cell culturemedium for the infection (i.e. during step c and/or step b).

According to a preferred embodiment, the cell culture media according tothe invention is free from animal product.

Many media free from animal product has been already described and someof them are commercially available. For example 293 SFM II; 293-F Cells,SFM Adapted; 293-H Cells, SFM Adapted; 293fectin™ Transfection Reagent;CD 293 ACT™; CD 293 Medium; FreeStyle™ 293 Expression System; FreeStyle™293 Medium; FreeStyle™ 293-F Cells, SFM Adapted; Adenovirus ExpressionMedium (AEM) Growth Medium for PER.C6® Cells; CD 293 AGT™; CD 293Medium; COS-7L Cells, SFM Adapted; EPISERF® Medium; OptiPro™ SFM;VP-SFM; VP-SFM AGT™. (all available from invitrogen) can be used as cellculture media in the process according to the invention.

When the packaging cell is a CEF, VP-SFM (invitrogen) for step a andBasal Medium Eagle (invitrogen) for step b and c are particularlypreferred.

In the specific embodiment where the packaging cells are CEF, the cellculture medium is seeded with between between 0.5 to 1.5 and preferablybetween 1.1 and 1.3 and more preferably about 1.2 embryo/l of cellculture medium. In this embodiment, the CEF are preferably cultivatedfor between 1 and 5 days, more preferably between 1 and 2 days and evenmore preferably 2 days before infection.

In the specific embodiment where the poxvirus to produce is MVA, thevirus is introduced in the cell culture vessel at a MOI which ispreferably comprised between 0.001 and 0.1, more preferably between 0.03and 0.07 and even more preferably about 0.05.

According to a preferred embodiment of the invention, during step c, thepackaging cells are cultivated at a temperature which is lower than 37°C., preferably between 30° C. and 36.5° C. or between about 32° C. andabout 36° C., more preferably between 33° C. and 35° C., most preferablyat 34° C.

According to preferred embodiment of the invention, step c lasts betweenone and six days, more preferably between two and four days and mostpreferably about 72 hours.

After the infection step, the cell culture media used to cultivate thepackaging cell is collected. Said cell culture media comprised the EEVparticles shed by the infected packaging cell. According to a preferredembodiment, after the infection step, the cell culture media and thepackaging cell are collected. The cell culture media and the packagingcells can be pooled or collected separately.

In order to recover the poxviruses present in the packaging cells, theprocess according to the invention may comprises a step allowing thedisruption of the packaging cell membrane. This step leads to theliberation of the poxvirus from the packaging cell. The disruption ofthe packaging cell membrane can be induced by various techniques wellknown by the one skilled in the art. These techniques comprised but arenot limited to sonication, freeze/thaw, hypotonic lysis andmicrofluidization.

According to a preferred embodiment of the invention, the packaging cellmembrane is disrupted by using a high speed homogenizer. High speedhomogenizers are commercially available from Silverson Machines Inc(East Longmeadow, USA) or Ika-Labotechnik (Staufen, Germany). Accordingto particularly preferred embodiment, said High Speed homogeneizer is aSILVERSON L4R.

When the packaging cell and the cell culture media are pooled, thedisruption of the packaging cell membrane is not induced by freeze/thawas this technique leads to the destruction of the EEV particles(Ichihashi y. et al., 1996, virology, 217(2), 478-85).

According to a preferred embodiment, step d) further comprises aclarification step allowing the withdrawal of the cellular debris.According to a more preferred embodiment of the invention, saidclarification step is a depth filtration step.

Depth filtration includes but are not limited to the use of one or morecommercially available products: CUNO Incorporated AP series depthfilters (Examples include AP01), CUNO Incorporated CP series depthfilters (Example include CP10, CP30, CP50, CP60, CP70, CP90), CUNOIncorporated HP series depth filters Examples include HP10, HP30, HP50,HP60, HP70, HP90), CUNO Incorporated Calif. series depth filters(Examples include CA10, CA30, CA50, CA60, CA70, CA90), CUNO IncorporatedSP series depth filters (Examples include SP10, SP30, SP50, SP60, SP70,SP90), CUNO Delipid and Delipid Plus filters, Millipore Corporation CEseries depth filters (Examples include CE15, CE20, CE25, CE30, CE35,CE40, CE45, CE50, CE70, CE75), Millipore Corporation DE series depthfilters (Examples include DE25, DE30, DE35, DE40, DE45, DE50, DE55,DE560, DE65, DE70, DE75), Millipore Corporation HC filters (Examplesinclude A1HC, B1HC, COHC), CUNO Polynet Filters (An example includePolynet-PB), Millipore Clarigard and Polygard filters, CUNO Life Assurefilters, ManCel Associates depth filters (Examples include but are notlimited to PR 12 UP, PR12, PR 5 UP), and PALL or SeitzSchenkIncorporated filters. In order to improve the clarification capacity ofthe available depth filtration units, it can be useful to couple two ormore units with decreasing pore sizes. In this embodiment, the mixtureto be clarified passes through the first depth filtration unit where thebiggest contaminants are retained and subsequently passes through thesecond depth filtration unit. According to an even more preferredembodiment of the invention, a sartopure© (sartorius) with a pore sizeof 8 μm coupled to a sartopure© with a pore size of 5 μm is used for thedepth filtration step.

According to a preferred embodiment, the process according to theinvention further comprises a concentration step. More preferably, saidconcentration step further allows the elimination of the proteinspresent in the mixture obtained from the previously described steps.

According to a more preferred embodiment of the invention, saidconcentration step is a microfiltration step. Microfiltration is apressure driven membrane process that concentrates and purifies largemolecules. More specifically, a solution is passed through asemi-permeable membrane whose pore sizes have been chosen to reject thelarge particles (viruses) in the retentate, and allow the smallmolecules (e.g. proteins) to pass through the membrane into thepermeate. Microfiltration reduces the volume of the extraction solution.

According to a preferred embodiment of the invention, themicrofiltration step is followed by a diafiltration step. These twosteps can advantageously be done with the same filtration membranes.Diafiltration is an improvement of microfiltration and involves dilutingthe retentate with a solution to effect a reduction in the concentrationof the impurities in the retentate. The dilution of the retentate allowswashing out more of the impurities from the retentate. It is understoodthat the diafiltration may be carried out in a batch mode,semi-continuous mode, or a continuous mode. The diafiltration step canbe advantageously used to change the buffer in which the virus iscomprised. For example, it can be useful to exchange the buffer used inthe purification process against a pharmaceutically acceptable buffer

Preferably, the filtration membranes used in the microfiltration and/orin the diafiltration step have a pore size comprised between 0.01 and0.15 μm, and more preferably about 0.1 μm.

Nucleic acids may adhere to cell derived particles such as viruses. Withthis respect, the process according to the invention can optionallyfurther comprises a step consisting in removing the contaminatingnucleic acids present in the solution. For this purpose, nucleases canbe utilized. Exemplary nucleases include Benzonase or any other DNase orRNase commonly used within the art, among them, Benzonase isparticularly preferred.

Benzonaze degrades nucleic acid and have no proteolytic activity.Benzonase rapidly disintegrates nucleic acid by hydrolyzing internalphosphodiester bonds between specific nucleotides. Upon completedigestion, all free nucleic acids present in solution are reduced tooligonucleotides 2 to 4 bases in length.

Benzonase can be used at a final concentration comprised between 50 and400 U/ml, preferably between 100 and 300 U/ml and more preferablybetween 150 and 250 U/ml. The Benzonase treatment can last between 1 and4 hours and even more preferably between 1.5 and 2.5 hours.

However, when the depth filtration, microfiltration and diafiltrationpreviously described are used, the use of nucleases and moreparticularly, the use of benzonase is not necessary. With this respect,the present invention also relates to a process for the production ofpoxviruses wherein no nuclease and more particularly no benzonase isused.

At this stage, the poxvirus obtained by the process previously describedis sufficiently purified. It can be desirable to change the buffer inwhich the virus is comprised. For example, it can be useful to exchangethe buffer used in the purification process against a pharmaceuticallyacceptable buffer. Many methods usable to change the buffer are known tothe one skilled in the art. Among them, tangential filtration isparticularly preferred.

Tangential filtration is a filtration technique wherein the suspensionto be filtered moves at a high speed along a direction which is parallelto filtrating surface in order to create a suspension turbulence whichprevents the filtration cake formation as well as the frequent filterblocking.

While suspension flows at high speed parallel to filtering surface,solute passes through the filtering surface holes because of thepressure difference and is continuously removed. Filtering surfaceshould be such as to mechanically resist to pressure difference betweenthe retentate compartment (i.e. the suspension concentratingcompartment) and the permeate compartment (clear filtered solution freefrom suspended particles).

The various steps comprised in the process according to the inventioncan be implemented in separated working places and/or in different timeperiods. For example, the infection of the packaging cell and thepurification of the poxviruses can be made in different working places.As well, the packaging cell and/or the cell culture media can be stored(e.g. at −80° C.) as long as necessary, before being purified.

The present invention also relates to compositions obtained by theprocess previously described and to composition comprising the poxvirusof the invention.

Preferably, the composition of the invention comprises more than 1%,preferably more than 5%, even more preferably more than 10% and mostpreferably at least 20% of the poxviruses comprised in said compositionare EEV.

According to a preferred embodiment, the composition according to theinvention has a titer of at least 10⁵, preferably of at least 10⁶, morepreferably of at least 10⁷, even more preferably of at least 10⁸ pfu perml.

According to another preferred embodiment, the composition according tothe invention has a titer of at least 10³, preferably 10⁴ and mostpreferably at least 3 10⁵ pfu per μg of protein.

The present invention also relates to pharmaceutical compositioncomprising the poxvirus obtained by the process of the invention, and/orcomposition according to the invention. As used herein, “pharmaceuticalcomposition” refers to a composition comprising a pharmaceuticallyacceptable carrier.

Such a carrier is preferably isotonic, hypotonic or weakly hypertonicand has a relatively low ionic strength, such as for example a sucrosesolution. Moreover, such a carrier may contain any solvent, or aqueousor partially aqueous liquid such as nonpyrogenic sterile water. The pHof the pharmaceutical composition is, in addition, adjusted and bufferedso as to meet the requirements of use in vivo. The pharmaceuticalcomposition may also include a pharmaceutically acceptable diluent,adjuvant or excipient, as well as solubilizing, stabilizing andpreserving agents. For injectable administration, a formulation inaqueous, nonaqueous or isotonic solution is preferred. It may beprovided in a single dose or in a multidose in liquid or dry (powder,lyophilisate and the like) form which can be reconstituted at the timeof use with an appropriate diluent.

The present invention also relates to the poxvirus and the compositionobtained by the process according to the invention.

The present invention also relates to the use of a process according tothe invention for the production of a virus, a composition and/or apharmaceutical composition.

The present invention also relates to the use of a poxvirus, acomposition and/or a pharmaceutical composition according to theinvention for the preparation of a medicament.

According to a preferred embodiment the medicament according to theinvention is for the therapeutic or prophylactic treatment of cancer.

Among the applications which may be envisaged, there may be mentionedcancers of the breast, of the uterus (in particular those induced bypapilloma viruses), of the prostate, of the lungs, of the bladder, ofthe liver, of the colon, of the pancreas, of the stomach, of theesophagus, of the larynx, of the central nervous system (in particularglyoma) and of the blood (lymphomas, leukemia and the like).

A composition according to the invention may be manufacturedconventionally for administration by the local, parenteral or digestiveroute. The routes of administration which may be envisaged are many.There may be mentioned, for example, the intragastric, subcutaneous,intracardiac, intramuscular, intravenous, intraperitoneal, intratumor,intranasal, intrapulmonary or intratracheal route. For the latter threeembodiments, administration by aerosol or instillation is advantageous.The administration may be made as a single dose or repeated once orseveral times after a certain time interval. The appropriate route ofadministration and dosage vary as a function of various parameters, forexample, of the individual, of the disease to be treated or of thegene(s) of interest to be transferred.

According to a first possibility, the medicament may be administereddirectly in vivo (for example by intravenous injection, into anaccessible tumor or at its periphery, subcutaneously for a therapeuticor prophylactic vaccination). It is also possible to adopt the ex vivoapproach which consists in collecting cells from the patient (bonemarrow stem cells, peripheral blood lymphocytes, muscle cells and thelike), transfecting or infecting them in vitro according to prior arttechniques and readministering them to the patient.

It is moreover possible to envisage, where appropriate and withoutdeparting from the scope of the present invention, carrying outsimultaneous or successive administrations, by different routes, of thevarious components contained in the pharmaceutical composition orcomposition according to the invention.

According to an advantageous embodiment of the invention, thetherapeutic use or the method of treatment is combined with a secondtreatment of the patient by surgery (partial or complete removal of thetumor), by radiotherapy or chemotherapy. In this particular case, thetreatment according to the invention is applied prior to, concomitantlywith or subsequent to said second treatment. Preferably, this treatmentwill be applied subsequent to said second treatment.

According to another preferred embodiment the medicament according tothe invention is for the therapeutic or prophylactic treatment ofinfectious diseases, in particular of viral origin, induced by hepatitisB or C viruses, HIV, herpes, retroviruses and the like

The examples which follow are intended to illustrate the varioussubjects of the present invention and are consequently not limiting incharacter.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: depicts the survival rate of C57BL/6 mice which as beenimplanted with TC1 cells expressing HPV 16 E6 and E7 and C-Ha-rasoncogene. The different groups of mice are treated with compositionscomprising IMV and EEV encoding HPV antigens, IMV encoding the HPVantigens or IMV encoding no antigens.

FIG. 2: depicts percentage of C57BL/6 mice free from tumor afterimplantation with TC1 cells expressing HPV 16 E6 and E7 and C-Ha-rasoncogene. The different groups of mice are treated with compositionscomprising IMV and EEV encoding HPV antigens, IMV encoding the HPVantigens or IMV expressing no antigens.

FIG. 3: depicts the survival rate of C57BL/6 mice which as beenimplanted with TC1 cells expressing MUC1. The different groups of miceare treated with compositions comprising IMV and EEV encoding MUC1 andIL2, IMV encoding MUC1 and IL2 or IMV expressing no antigens.

EXAMPLES 1. Preparation of Compositions comprising IMV and EEV

A. Preparation of CEF.

Sixty six SPF eggs are incubated in for 60s. in a 2% formol solution.After being rinsed with 70% ethanol, the eggs are open, the embryos areextracted and dissected. The obtained tissues are then digested at 36.5°C. for 120 min by dispase (UI/ml) and triple select (UI/ml).

The mixture is filtrated to remove undigested tissues and the CEF arecollected by centrifugation (2300 rpm, 15 min.)

B. CEF Cultivation and Infection.

The CEF are incubated in 55 I of VP-SFM (invitrogen) for 2 days at 36.5°C. The cell culture media is then discarded and the poxvirus (0.05 MODis added in 55 I of Basal Medium Eagle (invitrogen). The infectedpackaging cells are then incubated for three days.

C. Poxvirus Purification.

The packaging cell and the cell culture media are collected. The mixtureis then homogenised for 15 min. with a Silverson© L4R high speedhomogeniser. The obtained mixture is then clarified by depth filtrationon a sartopure 8 μm (sartorius) coupled to a sartopure 5 μm at a flowrate of 11/min.

The mixture is further concentrated 18 times through a 0.1 μm ProstakMicrofiltration Module (ref: PSVVAG021, Millipore).

The poxyiral composition is further diafiltrated on the same moduleagainst the desired pharmaceutically acceptable carrier.

2. Therapeutic Efficiency Comparison Between a Composition ComprisingEEV and IMV and a Composition Comprising IMV Only

A. Therapeutic Treatment of Mice Bearing Tumor Expressing HPV Antigens.

Denomination and Brief description of each Vector Construction

TG4001: MVA vector carrying the coding sequences for HPV proteins, E6and E7, (under the control of promoter p7.5) and IL2 (under the controlof promoter pH5R). Two lots, one comprising IMV and EEV (prepared aspreviously disclosed) and one comprising only IMV were tested.

N33: empty vector MVA which expresses neither HPV proteins E6 and E7 norIL2 was used as negative control.

Animal Model

C57B1/6 female mice aged 6 to 8 weeks were used throughout this study.These mice were obtained from Charles River (Rouen, France).

Specification

The animals were 6 week old on the arrival day. At the beginning ofexperimentation, they were less than 8 week old.

Environment

The animals were housed in a single, exclusive room air-conditioned toprovide a minimum of 11 air changes per hour. The temperature andrelative humidity ranges were within 18° C. and 22° C. and 40 to 70%respectively. Lighting was controlled automatically to give a cycle of12 hours of light and 12 hours of darkness.

Specific pathogen free status is checked by regular control of theenvironment.

Diet

Throughout the study the animals had access ad libitum to sterilizeddiet type D04 (UAR, Epinay sur Orge, France). Water was provided adlibitum via bottles.

Acclimatization and Health Procedures

All animals were given a clinical inspection for health on arrival. Theywere acclimatized in a specific pathogen free (SPF) animal facilitybetween one and two weeks before the start of the experiment in order toensure their suitability for the study.

Tumor Cells Characteristics and Conditions of Use:

The TC1 line has been derived from primary lung epithelial cells ofC57B1/6 mice co-transformed with HPV-16 E6 and E7 and c-Ha-rasoncogenes. These cells grow in DMEM with Glutamine (2 mM), fetal calfserum (10%), non essential amino acids (0.1 mM), Na Pyruvate (1 mM), βmercapto-ethanol (36 μM), Hygromycine (0.2 mg/ml) and G418 (0.5 mg/ml).After thawing, cells were amplified two times, the latest passage wasperformed two days before the cell injection.

Cells Injection

The first day of the experiment, TC1 cells were injected subcutaneouslyin mice at a dose of 2.0 E+05 cells/mouse in the flank.

Viral Injections

7 days after the cells injection, 5.0 E+05 pfu/50 μl/mouse of testedlots (TG4001 IMV/EEV or IMV only) MVATGN33 (empty vector) were injectedinto mice. Twenty mice were used per tested lot.

Viral injections were performed subcutaneously in the same flank but ata distant site from the cells injection point and carried out 3 times at7 day intervals.

Parameters of Monitoring:

Tumor growth was monitored for 90 days after the cells injection, with acaliper. Mice were sacrificed for ethical reasons when the tumor sizewas superior to 25 mm in diameter or when they showed pain even if thetumor was smaller.

Surviving mice were recorded.

Results

All the groups treated with the MVA vector encoding HPV antigen showed ahigher survival rate than the group treated with an empty MVA vector.The mice treated with the composition comprising EEV and IMV showed ahigher survival rate than the mice treated by composition comprising IMVonly. Moreover, 35% of the mice treated by the composition comprisingEEV and IMV were free from tumor 77 days after being injected comparedto only 10% of the mice treated by the composition comprising only IMV.

B. Therapeutic Treatment of Mice Bearing Tumor expressing MUC1.

Denomination and Brief Description of each Vector Construction

TG4010: MVA vector carrying the coding sequences for MUC1 proteins(under the control of promoter p7.5) and IL2 (under the control ofpromoter pH5R). Two lots, one comprising IMV and EEV and one comprisingonly IMV were tested.

An empty vector MVATGN33 which expresses neither MUC1, nor IL2 was usedas negative control.

Mouse model and animal experiments system

Species, Strain and Supplier

C57B1/6 female mice aged 6 to 8 weeks were used throughout this study.These mice were obtained from Charles River (Rouen, France).

Specification

The animals were 6-week old on the arrival day. At the beginning ofexperimentation, they were less than 8 week old.

Environment

The animals were housed in a single, exclusive room air-conditioned toprovide a minimum of 11 air changes per hour. The temperature andrelative humidity ranges were within 18° C. and 22° C. and 40 to 70%respectively. Lighting was controlled automatically to give a cycle of12 hours of light and 12 hours of darkness.

The animals were housed in-groups of 10 per cages of 43×27×15 cm, floorarea 1161 cm².

Diet

Throughout the study the animals had access ad libidum to sterilizeddiet type RM (SDS, Le Bord'Haut de Vigny, France).

Water was provided ad libidum via bottles.

No contaminants were present in diet or water at levels, which mighthave interfered with achieving the objectives of the study.

Acclimation and Health Procedures

All animals were given a clinical inspection for health on arrival. Theywere acclimatized in a Specific Pathogen Free (SPF) animal facilitybetween one and two weeks before the start of the experiment in order toensure their suitability for the study.

Tumor Cells Characteristics and Conditions of Use:

The RMA tumor line has been derived from a C57B1/6 lymphoma. RMA-MUC1cells were obtained after transfection with an expression plasmidcontaining the MUC1 gene a. These cells were grown in DMEM withGlutamine (2 mM), fetal calf serum (10%), non essential amino acids (0.1mM), Na Pyruvate (1 mM), β mercapto-ethanol (36 μM) and Hygromycine (550μg/ml). After thawing, cells were amplified two times, the latestpassage was performed the day before challenge.

Immunization

Mice were immunized with 1.0 104 or 3.0 104 pfu/mouse for TG4010 virusand with 3.0 104 pfu/mouse for MVATGN33.

20 mice were used per tested lot.

Viral immunizations were performed subcutaneously in a flank and carriedout 3 times at 14 day intervals.

Tumor Challenge

Two weeks after the last immunization, mice were challengedsubcutaneously in the same flank but at a distant site from the viralinjections point, with 1.0 E+06 RMA-MUC1 viable cells/50 μl/mouse.

Parameters of Monitoring:

Tumor growth was monitored for 6 weeks after the tumor challenge, with acaliper. Mice were sacrificed for ethical reasons when the tumor sizewas superior to 25 mm in diameter or when they showed pain even if thetumor was smaller.

Surviving mice were recorded.

20 mice were used per dose.

Results.

All the groups treated with the MVA vector encoding the MUC1 antigenshowed a lower tumor growth and a higher survival rate than the grouptreated with an empty MVA vector. The mice treated with the compositioncomprising EEV and IMV showed a lower tumor growth than the mice treatedby composition comprising only IMV.

What is claimed is:
 1. A process for purifying a poxvirus compositionfrom cell culture supernatant and cultured packaging cells infected witha wild type, an attenuated, and/or a recombinant poxvirus with notargeted infection specificity, comprising the steps of: a) recoveringpoxyiral particles from the cell culture supernatant and from culturedpackaging cells disrupted by high speed homogenizer, to obtain apoxyiral particle mixture, b) clarifying said poxyiral particle mixture,thereby removing cellular debris, wherein said clarifying comprisesdepth filtration, to obtain a clarified poxyiral particle mixture, c)concentrating said clarified poxyiral particle mixture, wherein saidconcentrating comprises microfiltration, to obtain a concentratedpoxyiral particle mixture, and d) diafiltrating said concentratedpoxyiral mixture to obtain a composition of a wild type, an attenuated,and/or a recombinant poxvirus with no targeted infection specificitywherein said process is free from animal products and does not usenuclease, and wherein said poxvirus composition comprises intracellularmature virus (“IMV”) and extracellular enveloped virus (“EEV”) with morethan 1% EEV.
 2. The process of claim 1, wherein said poxviruscomposition comprises more than 5% EEV.
 3. The process of claim 1 orclaim 2, wherein said process for producing said poxvirus compositionfurther comprises step e) of tangential filtration.
 4. The process ofclaim 1, wherein said packaging cell is chicken embryo fibroblast (CEF)prepared by digestion of embryo tissues with trypsin and dispase.
 5. Theprocess of claim 1, wherein said poxvirus poxyirux is an orthopoxvirus.6. The process of claim 5, wherein said orthopoxvirus is a Vacciniavirus.
 7. The process of claim 6, wherein said Vaccinia virus isModified Vaccinia Virus Ankara (MVA).
 8. The process of claim 7, whereinsaid MVA is selected from MAV575 (ECACC V00120707) and MVA-BN (ECACCV00083008).
 9. The process of claim 1, wherein microfiltration and/ordialfiltration step is/are performed on filtration membranes having apore size of between 00.1 μm and 0.15 μm.
 10. The process of claim 9,wherein said filtration membranes have a pore size of 0.1 μm.